Gastrointestinal Symptoms and Ethanol Metabolism in Alcoholics

Transcription

1 Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004), pp ( C 2004) Gastrointestinal Symptoms and Ethanol Metabolism in Alcoholics R. J. F. LAHEIJ, PhD,* M. VERLAAN, MSc,* M. G. H. VAN OIJEN, MSc,* M. S. DE DOELDER, MSc,* C. A. J. DEJONG, MD, PhD, and J. B. M. J. JANSEN, MD, PhD* Excessive alcohol intake frequently results in gastrointestinal discomfort. It is an empirical fact that the severity of gastrointestinal discomfort induced by alcohol abuse is subject to interindividual variation. The aim of this study was to determine whether genetic polymorphism in alcohol dehydrogenase 3 (ADH3) and cytochrome P450 2E1 (CYP2E1), important first-pass enzymes in the metabolism of ethanol, predispose to the development of gastrointestinal symptoms in alcoholics. Blood samples were obtained from 92 adult alcoholics admitted for detoxification. The samples were analyzed for genetic polymorphism in ADH3 and CYP2E1 by polymerase chain reaction followed by restriction fragment length polymorphism analyses. During an interview on the first day of hospital admission, patient characteristics and gastrointestinal symptoms in the week before admission were assessed. A total of 75 of 92 alcoholics (83%) reported symptoms: 66 patients had upper gastrointestinal symptoms (72%), 70 patients had lower gastrointestinal symptoms (76%), and 59 patients reported alarming symptoms (64%). Patients with gastrointestinal symptoms less often abused beer in comparison to those without gastrointestinal symptoms (P = 0.05). The numbers of patients with the homozygous γ 1γ 1 genotype, the heterozygous γ 1γ 2 genotype, and the homozygous γ 2γ 2 genotype in ADH3 who reported gastrointestinal symptoms were 20 (83%), 34 (76%), and 15 (88%), respectively. The number of patients with the heterozygous c1c2 CYP2E1 genotype (5%) and the heterozygous DC CYP2E1 genotype (14%) was low and also unrelated to gastrointestinal symptoms. Our data suggest that the ethanol concentrations of the consumed beverages, and not interindividual variations in the activities of first-pass alcohol-metabolizing enzymes, are associated with gastrointestinal symptoms in alcoholics. KEY WORDS: ethanol; gastrointestinal symptoms; metabolism; alcohol dehydrogenase; cytochrome P450 2E1. Ethanol consumption is considered to be an important risk factor for the development of gastrointestinal discomfort and complications (1). Side effects of ethanol consumption on upper and lower gastrointestinal tract are common. However, not every alcohol consumer develops discomfort or complications. Consequently, it might be possible that Manuscript received October 2003; accepted April 14, From the *Department of Gastroenterology, University Medical Center St. Radboud, and Novadic Network for Addiction Treatment Services/Institute for Scientists Practitioners in Addiction, University of Nijmegen, Nijmegen, The Netherlands. Address for reprint requests: Robert Laheij, University Medical Center St. Radboud, Department of Gastroenterology, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands; R.Laheij@mdl.umcn.nl. susceptibility could be influenced by interindividual variations in the activities of ethanol-metabolizing enzymes (2). Ethanol is metabolized predominantly by the liver, whereas 20% of ethanol metabolism may already occur in the gastric mucosa. Ethanol is detoxified in two steps during the first passage through the liver. The speed of every step is enzyme dependent. The enzymes involved with the first step are alcohol dehydrogenase 3 (ADH3) and cytochrome P450 2E1 (CYP2E1) (3 6). A reduction in first-pass metabolism accumulates the amount of ethanol for a longer period in the body. The local alcohol toxicity is mediated by acetaldehyde on the gastric mucosa. Acetaldehyde inhibits mucosal regeneration and forms stable adducts with mucosal proteins Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004) /04/ /0 C 2004 Plenum Publishing Corporation

2 LAHEIJ ET AL. causing gastric injury and is therefore being considered as a local carcinogen in the gastrointestinal tract. Excessive alcohol abusers are known to have more chronic gastritis than moderate or non-alcohol users (10). Persons consuming excessive amounts of alcohol with the rapid metabolizing alcohol dehydrogenase genotype have a greater risk for upper gastrointestinal malignancies in comparison to persons with other genotypes (11). The rate of ethanol metabolism variability is genetic in origin and varies between racial populations. The ADH3 gene carries two alleles, ADH3*1 and ADH3*2, which differ by one amino acid, and encodes the subunits γ 1 and γ 2, respectively. The isoenzyme γ 1 γ 1 metabolizes ethanol 2.5-fold as fast as the γ 2 γ 2 isoenzyme (4). The CYP2E1 enzyme particularly contributes to ethanol metabolism in chronic alcohol consumers. Genetic polymorphisms in CYP2E1 have been demonstrated in the transcriptionregulation region (alleles c1 and c2) and in intron 6 (alleles D and C) (5). The presence of the c2 or C allele was associated with enhanced enzyme activity (5, 6). The aim of this study was to determine whether polymorphism in the genes for ADH3 and CYP2E1 is associated with the development of gastrointestinal symptoms in alcoholics. METHODS Consecutive patients were recruited between January and November 2002 at four detoxification clinics. Patients were included if they have reached the age of 18 and were ethanol dependent following DSM-IV criteria. Patients were asked to participate on the first day of admission. After written informed consent, blood samples were taken and stored in EDTA tubes at 20 C. Patients were interviewed to assess the frequency of gastrointestinal symptoms in the week before admission. The interview included inquiries on the presence of the following gastrointestinal symptoms: abdominal pain, abdominal distension, epigastric pain, heartburn, regurgitation, bloating, flatulence, nausea, dysphagia, weight loss, hematemesis, meleana, colorectal bleeding, and diarrhea. Symptoms were scored on a 4-point Likert scale: none, mild (can be ignored), moderate (cannot be ignored but does not affect lifestyle), and severe (affects lifestyle). Serological screening for Helicobacter pylori infection was performed with the Pyloriset, which was locally validated (Pyloriset test kit; Imphos, the Netherlands). The Pyloriset is a commercially available enzyme-linked immunosorbent assay that measures IgG antibodies to H. pylori infection. Furthermore, patient characteristics and ethanol consumption-related information were collected. The ethics committee of the hospital approved the study protocol. Genotyping. DNA was isolated from whole blood using the Pure Gene DNA isolation kit, and isolation was performed according to the instructions of the manufacturer (Gentra systems). The genotyping for the polymorphism in ADH3 was carried out by polymerase chain reaction (PCR) amplification according to the method of Xu et al. (7). Subsequently the PCR product was digested using the restriction enzyme SspI (PCR/RFLP). The ADH3*1 allele contains an additional restriction site for SspI, yielding fragments of 63 and 67 bp, which is not present in the ADH3*2 allele. The CYP2E1 genotypes were determined by PCR/RFLP methods as described elsewhere (8). The restriction enzymes RsaI and PstI were used to discern the c1 and c2 alleles of the 5 -flanking region, while the enzyme DraI was used to detect the D and D polymorphisms in intron 6. Analyses. We estimated that 50% of the patients with the γ 2 γ 2 slower metabolizing genotype and 80% of the patients with the γ 1 γ 1 or γ 1 γ 2 rapid metabolizing genotype would report gastrointestinal symptoms. To detect the difference of 30% with a power of 0.80, an α of 0.05 (one-sided), and taking into account the unequal distribution of ADH3 polymorphism (a ratio of 0.3), a total of 90 patients were required. Chi-square and t-test statistics were used to estimate differences in baseline characteristics among patients with and without gastrointestinal symptoms. Abdominal pain, abdominal distension, and diarrhea were classified as lower gastrointestinal symptoms; epigastric pain, heartburn, regurgitation, bloating, flatulence, and nausea, as upper gastrointestinal symptoms. Dysphagia, weight loss, hematemesis, meleana, and colorectal bleeding were classified as alarming symptoms. Logistic regression analysis was performed to study the association between polymorphism and upper gastrointestinal symptoms, lower gastrointestinal symptoms, and alarming symptoms in order to estimate an odds ratio with 95% confidence interval (CI) for these variables. Because other factors besides interindividual variations in the activities of ethanol-metabolizing enzymes might be related to the presence of gastrointestinal symptoms, we performed a multivariate regression analysis to adjust for acid secretion inhibitory therapy and H. pylori infection. Data were analyzed by SAS version 8.0. RESULTS We included 92 Caucasian patients in the study; 73 were male and the average age was 42 years. The patients reported on average a daily ethanol consumption of 200 g for more than 10 years. A total of 75 of 92 patients (83%) suffered from gastrointestinal symptoms: 66 reported upper gastrointestinal symptoms (72%), 70 reported lower gastrointestinal symptoms (76%), and 59 patients had symptoms suspiciious for a malignancy (64%) (Table 1). Thirty-seven patients reported their alarming symptoms as mild, 26 as moderate, and 3 as severe. Patients with gastrointestinal symptoms less often abused beer in comparison to those without gastrointestinal symptoms (P = 0.05). All other patient characteristics were similar between patients with and those without gastrointestinal symptoms (Table 2). Many patients (39%) were using acid-suppressive drugs to control gastrointestinal symptoms. From patients using medication to control the gastrointestinal symptoms, 31% reported complete symptom relief, while in the other 69% symptoms did not disappear. ADH3 Genotyping. In 86 of the 92 patients we were able to evaluate the ADH3 genotype. The number of patients with the homozygous γ 1γ 1 genotype, the heterozygous γ 1γ 2 genotype, and the homozygous γ 2γ Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004)

3 GI SYMPTOMS AND ETHANOL METABOLISM IN ALCOHOLICS TABLE 1. FREQUENCY OF GASTROINTESTINAL SYMPTOMS IN THE WEEK BEFORE ADMISSION Number (%) None Mild Moderate Severe Overall 17 (18) 33 (36) 36 (39) 6 (7) Upper gastrointestinal symptoms 26 (28) 39 (42) 23 (25) 4 (4) Epigastric pain 63 (68) 20 (22) 8 (9) 1 (1) Heartburn 53 (58) 22 (24) 17 (19) 0 Regurgitation 52 (57) 28 (30) 10 (11) 2 (2) Bloating 46 (50) 28 (30) 15 (16) 3 (3) Nausea 54 (59) 27 (29) 11 (12) 0 Alarm symptoms 33 (36) 37 (40) 19 (21) 3 (3) Dysphagia 71 (77) 16 (17) 3 (3) 2 (2) Weight loss 58 (63) 20 (22) 13 (14) 1 (1) Hematemesis 85 (92) 5 (5) 2 (2) 0 Meleana 75 (82) 13 (14) 4 (4) 0 Colorectal bleeding 79 (86) 11 (12) 1 (1) 1 (1) Lower gastrointestinal symptoms 22 (24) 41 (45) 26 (28) 3 (3) Abdominal pain 57 (62) 33 (36) 2 (2) 0 Abdominal distension 35 (38) 33 (36) 22 (24) 2 (2) Diarrhea 49 (53) 32 (35) 10 (11) 1 (1) genotype was 24 (30%), 45 (52%), and 17 (20%), respectively (Table 3). There were no statistical differences in the distribution of the homozygous γ 1γ 1 genotype, the heterozygous γ 1γ 2 genotype and the homozygous γ 2γ 2 genotype among patients with upper gastrointestinal symptoms, lower gastrointestinal symptoms and alarming symptoms in comparison to patients without these symptoms. Even after adjustment for H. pylori infection and acid-suppressive therapy no association was found. TABLE 3. GASTROINTESTINAL SYMPTOMS AND ADH3 POLYMORPHISM ADH3, number (%) γ 1γ 1 γ 1γ 2 γ 2γ 2 Gastrointestinal symptoms (n = 24) (n = 45) (n = 17) Any Absent 4 (24) 11 (65) 2 (12) Present 20 (29) 34 (49) 15 (22) Upper Absent 7 (28) 13 (52) 5 (20) Present 17 (29) 32 (51) 12 (20) Lower Absent 5 (23) 12 (54) 5 (23) Present 19 (30) 33 (51) 12 (19) Alarm Absent 8 (25) 21 (66) 3 (9) Present 16 (30) 24 (44) 14 (26) CYP2E1 Genotyping. In 78 of the 92 patients included we were able to evaluate CYP2E1 genotypes. The number of patients with the homozygous c1c1 genotype, the heterozygous c1c2 genotype, and the homozygous c2c2 genotype was 74 (95%), 4 (5%), and 0 (0%), respectively. The number of patients with the homozygous DD genotype, the heterozygous DC genotype, and the homozygous CC genotype was 67 (86%), 11 (14%), and 0 (0%), respectively. Despite the fact that 10 of the 11 patients (91%) with the heterozygous DC CYP2E1 genotype reported upper gastrointestinal symptoms, 9 lower gastrointestinal symptoms (82%), and 8 alarming symptoms (73%), no statistical association was found between the CYP2E1 genotype and symptom clustering. TABLE 2. PATIENT CHARACTERISTICS BY GASTROINTESTINAL SYMPTOMS Symptoms Absent Present (n = 17) (n = 75) Male/female (No.) 14/3 57/18 Mean age (years) Helicobacter pylori seropositive (%) Smoking (%) Coaddiction Opiates (%) Cocaine (%) Cannabis (%) Medication H2-RA (%) 6 8 PPI (%) 6 19 Alcohol intake (g ethanol/day) Mean addiction duration (years) Previous detoxification (%) Beverage Beer (%)* Wine (%) Liquor (%) 7 10 *P = DISCUSSION Gastrointestinal symptoms and complications associated with ethanol abuse are common. The results from this study confirm that persons abusing ethanol often have gastrointestinal symptoms; even alarming symptoms were frequently reported. Gastrointestinal symptoms were significantly related to non-beer abusers. Our study was designed to evaluate whether interindividual variations in the activities of ethanol-metabolizing enzymes were associated with gastrointestinal symptoms. However, interindividual variations in the activities of ADH3 and CYP2E1 enzymes were not associated with gastrointestinal symptoms. This finding is, however, in line with the fact that symptoms are poorly correlated with the severity of mucosal injury. Furthermore, because most patients reported persistent symptoms during acid-suppressive therapy, the impact of medical management of gastrointestinal symptoms in alcoholics on symptom improvement seems minimal. Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004) 1009

4 LAHEIJ ET AL. The only positive finding of this study was a significant negative correlation between gastrointestinal symptoms and beer drinking. This is most likely because gastrointestinal disturbances depend on the ethanol concentration of the consumed beverages and are due either to a direct effect of highly concentrated ethanol or to the activity of enzymes with a high K m for ethanol known to prevail in the upper digestive tract, such as σ -ADH (a class IV ADH), which plays no role at systemic concentrations of ethanol but could at the very high concentrations of ethanol achieved in the upper digestive tract after drinking. The role of the two enzymes we have investigated is minimal in some of the organs where the upper gastrointestinal symptoms may be generated. The principal catalyst of ethanol in the gastroesophageal area is ADH7, a class IV alcohol dehydrogenase (σ -ADH). However, so far no major polymorphisms of the ADH7 gene have been found, except for a point mutation (16). The frequency of this point mutation is very low in the Caucasian population and therefore it is probably not responsible for the development of gastrointestinal symptoms. Alcohol dehydrogenase is an important first-step enzyme in the oxidation of alcohol to acetaldehyde (4, 9). Especially, ADH3 is the most important enzyme in the oxidation of ethanol to acetaldehyde in Caucasians. However, oxidation of alcohol by the CYP2E1 enzymes is also important, especially in chronic alcohol abusers, as it is inducible up to 10-fold by consumption of alcohol. Only few patients were heterozygous for the CYP2E1 genotype. To detect a difference in frequency of genotype between patients with and those without symptoms, many more patients need to be included. However, CYP2E1 is probably not an important risk factor for gastrointestinal symptoms because few patients are heterozygous for its genotypes. Furthermore, taking into consideration that most patients in our study had been abusing alcohol for more than 10 years, there might be an enhancement of induction of increased enzyme metabolism activity after long-term alcohol intake. Several other factors may be related to the occurrence of gastrointestinal symptoms. For example, Helicobacter pylori infection leads to morphologic gastric mucosa changes which might be associated with a decrease in alcohol metabolism (12 14). It is, however, unclear whether this reflects the bioavailability of alcohol or the amount of its first-pass metabolism. In our study, however, we did not find an association between symptoms and Helicobacter pylori infection. This finding might be a consequence of the limited number of alcoholics in our study. Despite the high prevalence of gastrointestinal symptoms, it is not known whether drugs for management of dyspeptic symptoms in the general population are equally effective in alcoholics. The impact of medical management of gastrointestinal symptoms in alcoholics seems to be minimal. Most patients using drugs for upper gastrointestinal symptoms have persistent symptoms. Traditionally, patients who present with gastrointestinal discomfort are being managed with acid-suppressive medication. Acid-suppressive medication might even increase symptoms. Treatment with proton pump inhibitors increases acetyldehyde production from ethanol (15). Furthermore, previous studies have shown that H2 receptor antagonists, especially cimitidine, are associated with increased blood alcohol levels by an effect on gastric alcohol dehydrogenase activity (3). The results from this study showed that despite many of the patients using gastric acid inhibitors for their upper gastrointestinal symptoms, many failed to respond adequately. Because of the poor results with acidsuppressive therapy, other drugs or management strategies to prevent gastrointestinal discomfort should be evaluated. In conclusion, more than three-quarters of these alcoholics who had on average abused ethanol for more than 10 years reported gastrointestinal symptoms. The combination of excessive drinking and the ADH3 and CYP2E1 genotypes was not associated with gastrointestinal symptoms. ACKNOWLEDGMENTS We would like to thank T. Wijdeveldt, MD, and P. Geenen from Paschalis, K. Rijk, MD, from the Grift, and H. de Haan, MD, from Tractus for helping collect the data. REFERENCES 1. Bujanda L: The effects of alcohol consumption upon the gastrointestinal tract. Am J Gastroenterol 95: , Dohmen K, Baraona E, Ishibashi et al.: Ethnic differences in gastric sigma-alcohol dehydrogenase activety and first-pass metabolism. Alcohol Clin Exp Res 20: , Haber PS, Gentry RT, Mak KM, Mirmiran Yazdy SAA, Greenstein RJ, Lieber CS: Methabolism of alcohol by human gastric cells: relation to first pass metabolism. Gastroenterology 111: , Bosron WF, Li TK: Genetic polymorphism of human liver alcohol and aldehyde dehydrogenases and their relationship to alcohol metabolism and alcoholism. Hepatology 6: , Hayasashi S, Watanabe J, Kawajiri K: Genetic polymorphisms in the 5 -flanking region change transcriptional regulation of the human cytochrome P-450IIE1 gene. J Biochem 110: , Watanabe J, Hayashi S, Kawajiri K: Different regulation and expression of the human CYP2E1 gene due to the RsaI polymorphism in the 5 -flanking region. J Biochem 16: , Xu YL, Carr LG, Bosron WF, Li TK, Edenberg HJ: Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification. Genomics 2: , Stephens EA, Taylor JA, Kaplan N, Yang CH, Hsieh LL, Lucier GW, Bell DA: Ethnic variation in the CYP2E1 gene: Polymorphism 1010 Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004)

5 GI SYMPTOMS AND ETHANOL METABOLISM IN ALCOHOLICS analysis of 695 African-Americans, European-Americans and Taiwanese. Pharmacogenetics 4: , Seitz HK, Poschl G: The role of gastrointestinal factors in alcohol metabolism. Alcohol Alcohol 32: , Hauge T, Persson J, Kjerstadius T: Helicobacter pylori, active chronic antral gastritis, and gastrointestinal symptoms in alcoholics. Alcohol Clin Exp Res 18: , Yokoyama A, Kato H, Yokoyama T, et al.: Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S- transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma. Carcinogenesis 23: , Lieber CS: Gastritis in the alcoholic: Relationship to gastric alcohol metabolism and Helicobacter pylori. Addict Biol 3: , Simanowski UA, Egerer G, Oneta C, et al.: Helicobacter pylori infection decreases gastric alcohol dehydrogenase activety and firstpass metabolism of ethanol in man. Digestion 59: , Lieber CS: Gastric ethanol methabolism and gastritis: Interactions with other drugs, Helicobacter pylori and antibiotic therapy. A review. Alcohol Clin Exp Res 21: , Vakevainen S, Tillonen J, Salaspuro M, Jousimies-Somer H, Nuutinen H, Farkkila M: Hypochlorhydria induced by a proton pump inhibitor leads to intragastric microbial production of acetyldehyde from ethanol. Aliment Pharmacol Ther 14: , Carr LG, Zeng D, Li TK: Failure to find exon 7 polymorphism of the ADH7 gene in Chinese, Japanese, African-Americans and Caucasians. Alchol Clin Exp Res 20: , 1996 Digestive Diseases and Sciences, Vol. 49, No. 6 (June 2004) 1011